The sphingolipids are a family of membrane lipids derived from the aliphatic amino alcohol sphingosine and its related sphingoid bases. They are present in eukaryote membranes, where they exert important structural roles in the regulation of fluidity and subdomain structure of the lipid bilayer. In addition, they have emerged as key effectors in many aspects of cell biology including inflammation, cell proliferation and migration, senescence and apoptosis [Hannun Y A, Obeid L M. Principles of bioactive lipid signalling: lessons from sphingolipids. Nat. Rev. Mol. Cell Biol. 2008, 9, 139-150]. Ceramide is considered a central molecule in sphingolipid catabolism. The generic term “ceramide” comprises a family of several distinct molecular species deriving from the N-acylation of sphingosine with fatty acids of different chain length, typically from 14 to 26 carbon atoms. Ceramide can be synthesized de novo from condensation of serine with palmitate, catalyzed by serine palmitoyltransferase, to form 3-keto-dihydrosphingosine. In turn, 3-keto-dihydrosphingosine is reduced to dihydrosphingosine, followed by acylation by a (dihydro)-ceramide synthase. Ceramide is formed by the desaturation of dihydroceramide. Alternatively, ceramide can be obtained by hydrolysis of sphingomyelin by sphingomyelinases. Ceramide is metabolized by ceramidases to yield sphingosine and fatty acid [Hannun Y A, Obeid L M, Nat. Rev. Mol. Cell Biol. 2008, 9, 139-150]. Ceramide plays an important role in a variety of cellular processes. Ceramide concentrations increase in response to cellular stress, such as DNA damage, exposure to cancer chemotherapeutic agents and ionizing radiation, and increased ceramide levels can trigger senescence and apoptosis in normal cells [Wymann M P, Schneiter R. Lipid signalling in disease. Nat. Rev. Mol. Cell. Biol. 2008, 9, 162-176]. Moreover, ceramide is also involved in the regulation of cancer cell growth, differentiation, senescence and apoptosis [Morad S and Cabot M. Ceramide-orchestrated signaling in cancer cells. Nat. Rev. Cancer 2013, 13, 51-65; Ogretmen B and Hannun Y A. Biologically active sphingolipids in cancer pathogenesis and treatment. Nat. Rev. Cancer 2004, 4, 604-616]. Many anticancer drugs increase ceramide levels in cells by stimulating its de novo synthesis and/or hydrolysis of sphingomyelin. For example, daunorubicin elicits ceramide production through the de novo pathway [Bose R et al., Ceramide synthase mediates daunorubicin-induced apoptosis: an alternative mechanism for generating death signals. Cell 1995, 82, 405-414]. De novo ceramide induction was observed in various human cancer cells after treatment with camptothecin and fludarabine [Chauvier D et al. Ceramide involvement in homocamptothecin-and camptothecin induced cytotoxicity and apoptosis in colon HT29 cells. Int. J. Oncol. 2002, 20, 855-863; Biswal 55 et al., Changes in ceramide and sphingomyelin following fludarabine treatment of human chronic B-cell leukemia cells. Toxicology 2000, 154, 45-53], and with gemcitabine [Chalfant C E et al., De novo ceramide regulates the alternative splicing of caspase 9 and Bcl-x in A549 lung adenocarcinoma cells. Dependence on protein phosphatase-1. J. Biol. Chem. 2002, 277, 12587-12595]. In many of these studies, inhibition of de novo ceramide synthesis was found to prevent, at least in part, the cytotoxic responses to these agents, thus indicating that the de novo pathway might function as a common mediator of cell death. Therefore, increasing or sustaining the levels of ceramide in cancer cells could be envisaged as a novel therapeutic strategy to induce cancer cell death.
One approach to increase or sustain the levels of ceramide in cells is to inhibit the enzymes responsible for ceramide clearance. Enzymes that contribute to decreasing the intracellular levels of ceramide are glucosylceramide synthase, which incorporates ceramide into glucosylceramide, sphingomyelin synthase, which synthesizes sphingomyelin, and ceramidases, which hydrolyze ceramide to sphingosine and fatty acid. Currently, there are five known human ceramidases: acid ceramidase (AC), neutral ceramidase, alkaline ceramidase 1, alkaline ceramidase 2, and alkaline ceramidase 3 [Mao C, Obeid L M. Ceramidases: regulators of cellular responses mediated by ceramide, sphingosine, and sphingosine-1-phosphate. Biochim. Biophys. Acta 2008, 1781, 424-434]. Among them, acid ceramidase is emerging as an important enzyme in the progression of cancer and in the response to tumor therapy [Gangoiti P et al., Control of metabolism and signaling of simple bioactive sphingolipids: Implications in disease. Prog. Lipid Res. 2010, 49, 316-334]. Messenger RNA and protein levels of acid ceramidase are heightened in a wide variety of cancers including prostate cancer [Seelan R S et al., Human acid ceramidase is overexpressed but not mutated in prostate cancer. Genes Chromosomes Cancer 2000, 29, 137-146], head and neck cancer [Norris J S et al., Combined therapeutic use of AdGFPFasL and small molecule inhibitors of ceramide metabolism in prostate and head and neck cancers: a status report. Cancer Gene Ther. 2006, 13, 1045-1051; Elojeimy S et al., Role of acid ceramidase in resistance to FasL: therapeutic approaches based on acid ceramidase inhibitors and FasL gene therapy. Mol. Ther. 2007, 15, 1259-1263], and melanoma [Musumarra G et al., A bioinformatic approach to the identification of candidate genes for the development of new cancer diagnostics. Biol. Chem. 2003, 384, 321-327]. In prostate cancer, acid ceramidase expression correlates with the malignant stage of the disease [Seelan R S et al., Human acid ceramidase is overexpressed but not mutated in prostate cancer. Genes Chromosomes Cancer 2000, 29, 137-146]. Up-regulation of acid ceramidase has also been observed in prostate cancer cells in response to radiotherapy, and this mechanism desensitizes cells to both chemotherapy and radiotherapy. Restoration of acid ceramidase levels in radio-resistant cells by either gene silencing or inhibition of acid ceramidase activity confers radiation sensitivity to prostate cancer cells. Improvement of tumor sensitivity to ionizing radiation by inhibition of acid ceramidase has been shown in vivo in a PPC-1 xenograft model [Mandy A E et al., Acid ceramidase upregulation in prostate cancer cells confers resistance to radiation: AC inhibition, a potential radiosensitizer. Mol. Ther. 2009, 5 17, 430-438]. Together, these data suggest that acid ceramidase provides a growth advantage to cancer cells and contributes to the altered balance between proliferation and death eventually leading to tumor progression. Therefore, inhibition of acid ceramidase appears to be a promising strategy for cancer treatment.
The aforementioned balance between cellular proliferation and death is mainly regulated by the ceramide/sphingosine 1-phosphate S1P rheostat [Mao C, Obeid L M. Ceramidases: regulators of cellular responses mediated by ceramide, sphingosine, and sphingosine-1-phosphate. Biochim. Biophys. Acta 2008, 1781, 424-434]. Compelling evidences implicate this pathway as contributor to inflammatory conditions and pain of diverse etiologies [Salvemini D, Doyle T, Kress M, Nicol G. Therapeutic targeting of the ceramide-to-sphingosine 1-phosphate pathway in pain. Trends in Pharmacological Sciences 2013, 34(2)110-118. Patti G J, Yanes O, Shriver L, Courade J P, Tautenhahn R, Manchester M, Siuzdak G. Metabolomics implicates altered sphingolipids in chronic pain of neuropathic pain. Nat Chem Biol 2013, 8(3), 232-234]. Blocking acid ceramidase implies an upstream inhibition of ceramide to sphingosine 1-phosphate S1P pathway and, therefore, seems to be a promising approach to inflammatory and pain conditions treatment.
Certain methods for inhibiting ceramidase activity by compounds containing a sphingoid base, a derivative of a sphingoid base, or a salt of a sphingoid base are described in the EP1287815. Other methods for inhibiting ceramidase activity using cyclopropenyl-sphingosine derivatives are described in WO2005/051891. Still other methods for inhibiting ceramidase activity in cells using cationic ceramide derivatives are reported in WO2006/050264. Further methods for inhibiting or modulating acid ceramidase activity are disclosed in WO2007/136635 and WO2010/054223. Acid ceramidase inhibitors disclosed in the scientific and patent literature, such as B13 [Selzner M et al., Induction of apoptotic cell death and prevention of tumor growth by ceramide analogues in metastatic human colon cancer. Cancer Res. 2001, 61, 1233-1240], D-e-MAPP [Bielawska A et al., (1S,2R)-D-Erythro-2-(N-myristolamino)-1-phenyl-1-propanol as an inhibitor of ceramidase. J. Biol. Chem. 1996, 271, 12646-12654], B13 and D-MAPP analogues [Proksch et al., Potent Inhibition of Acid Ceramidase by Novel B-13 Analogues, J. Lipids, Article ID 971618, 8 pages; Bielawska A et al., Novel analogs of D-e-MAPP and B13. Part 2: Signature effects on bioactive sphingolipids, Bioorg. Med. Chem. 2008, 16, 1032-1045; Szulc Z et al., Novel analogs of D-e-MAPP and B13. Part 1: Synthesis and evaluation as potential anticancer agents. Bioorg. Med. Chem. 2008, 16, 1015-1031; Bhabak K P and Arenz C, Novel amide-and sulfonamide-based aromatic ethanolamines: Effects of various substituents on the inhibition of acid and neutral ceramidases, Bioorg. Med. Chem. 2012, 20, 6162-6170], oleoylethanolamides such as NOE and NOE analogues [Grijalvo S et al., Design, synthesis and activity as acid ceramidase inhibitors of 2-oxooctanoyl and N-oleoylethanolamine analogues, Chem. Phys. Lipids 2006, 144, 69-84], LCL-204 [Holman D H et al., Lysosomotropic acid ceramidase inhibitor induces apoptosis in prostate cancer cells. Cancer Chemother. Pharmacol. 2008, 61, 231-242,], LCL-464 and analogues [Bai A et al., Synthesis and bioevaluation of omega-N amino analogs of B13, Bioorg. Med. Chem. 2009, 17, 1840-1848; Bhabak K P et al., Effective inhibition of acid and neutral ceramidases by novel B-13 and LCL-464 analogues, Bioorg. Med. Chem. 2013, 21, 874-882], or E-tb [Bedia C et al., Cytotoxicity and acid ceramidase inhibitory activity of 2-substituted aminoethanol amides. Chem. Phys. Lipids 2008, 156, 33-40] are ceramide analogs that inhibit acid ceramidase activity in cell-free assays and proliferation of cancer cell lines only at high micromolar concentrations.
However, recently, two different small-molecule chemotypes were reported to be acid ceramidase inhibitors—quinolinones [Draper J M et al., Discovery and Evaluation of Inhibitors of Human Ceramidase, Mol. Cancer Ther. 2011, 10, 2052-2061] and 2,4-dioxopyrimidine-1-carboxamides [Realini N et al., Discovery of highly potent acid ceramidase inhibitors with in vitro tumor chemosensitizing activity, Sci. Rep. 2013, 3, 1035; Pizzirani D et al., Discovery of a New Class of Highly Potent Inhibitors of Acid Ceramidase: Synthesis and Structure-Activity Relationship (SAR), J. Med. Chem. 2013, 56, 3518-3530] that have been disclosed in WO2013/178545 and WO2013/178576.
Although both series showed effects in vitro and in vivo, they may suffer from developability issues; therefore, there is a substantial need for novel acid ceramidase inhibitors with improved potency and drug-likeness, in particular selectivity over related proteins and intrinsic stability.
Certain benzoxazolonyl ureas are disclosed in FR1469297 for their fungicide, herbicide and pesticide properties and, more in general, as disinfectants of general use. Other benzoxazolones containing the urea moiety and their compositions are disclosed in FR2478635 as fungicides.
Finally, U.S. Pat. No. 7,709,513B2 discloses benzoxazol-2-one derivatives as lipase and phospholipase inhibitors. This reference only discloses that certain benzoxazol-2-one derivatives are active in metabolic diseases such as atherosclerosis and dyslipidemia and merely claims the treatment of insulin resistance and diabetes mellitus.